1. Field of the Invention
The present invention relates to improved receptor binding assays for the determination of TSH receptor autoantibodies (TSH-auto-ab) which occur in thyroid autoimmune diseases, in particular in Graves' disease.
2. Description of the Related Art
It is known that numerous diseases in which the thyroid is involved are autoimmune diseases in which autoantibodies against molecular structures of the thyroid are formed and, in association with the disease, begin to act as autoantigens. The most important known autoantigens of the thyroid are thyroglobulin (Tg), thyroperoxidase (TPO) and in particular the TSH receptor (TSHR) (cf. Furmaniak J et al., Autoimmunity 1990, Vol. 7, pages 63–80).
The TSH receptor is a receptor which is localized in the thyroid membrane and to which the hormone TSH (thyroid-stimulating hormone or thyrotropin) secreted by the pituitary gland binds and thus induces the secretion of the actual thyroid hormones, in particular of thyroxine. The TSH receptor belongs to the receptor family comprising the G-protein-coupled glycoprotein receptors with a large amino-terminal extracellular domain, to which the LH/CG receptor and the FSH receptor also belong. The chemical structure of the TSH receptor, i.e. of the sequence of the DNA coding for it and for the amino acid sequence, derivable therefrom, of the receptor itself, was determined at the end of 1989 (cf. Libert F. et al., Biochem. Biophys. Res. Commun. 165: 1250–1255; Nagayama Y. et al., Biochem. Biophys. Res. Commun. 165: 1184–1190; cf. also EP-A-0433509 or WO-A-91/09121; and WO-A-91/09137; WO-A-91/10735 and WO-A-91/03483; furthermore Yuji Nagayama & Basil Rapoport, in: Molecular Endocrinology, Vol. 6 No. 2, pages 145–156, and the literature cited therein).
It is generally known that stimulating autoantibodies which are formed against the TSH receptor and interact with it so that the thyroid is stimulated play a role in the thyroid autoimmune disease known as Graves' disease, which is manifested as thyroid hyperfunction (hyperthyroidism). The determination of autoantibodies against the TSH receptor is thus of considerable clinical importance for the diagnosis of Graves' disease.
TSHR-auto-ab have been determined to date in biological samples essentially by methods based on two principles (cf. for example Morgenthaler N. G. et al., J Clin Endocrinol Metab 81: 3155–3161 (1966)):
In cell stimulation tests, the presence of stimulating TSHR-auto-ab which are frequently denoted in the literature by the abbreviation TSI (TSI=thyroid stimulating immunoglobulins), is manifested by the fact that specific functions of suitable cells which have natural or recombinant TSHR in their cell membrane and come into contact with an autoantibody-containing sample are triggered or enhanced by stimulation, in particular the formation of cAMP (cyclic adenosine monophosphate). In these tests also referred to as bioassays, the stimulating effect is measured selectively but the measurement is extremely complicated and therefore not very suitable for routine clinical diagnosis.
Alternatively, autoantibodies can also be determined using competitive receptor binding assays, in particular radioreceptor assays, for example with the use of the TRAK-Assay® from B.R.A.H.M.S Diagnostica GmbH. For the determination of TSH receptor autoantibodies (TSHR-auto-ab), this conventional method is used in such a way that the autoantibodies to be determined and originating from a serum sample are allowed to compete in the liquid phase with a radiolabelled bovine TSH (125I-bTSH) for the binding sites of a solubilized porcine TSH receptor (porc. TSHR) (cf. Southgate, K. et al., Clin. Endocrinol. (Oxford) 20, 539–541 (1984); Matsuba T. et al., J. Biochem. 118, pages 265–270 (1995); EP 719 858 A2; product information on the TRAK-Assay® from B.R.A.H.M.S Diagnostica GmbH). To determine the 125I-bTSH bound to the porc. TSHR preparation, the solubilized porc. TSHR used is separated from the liquid phase by means of a precipitating reagent and subsequent centrifuging step after the incubation is complete. The receptor-bound 125I-bTSH is determined by measuring the radioactivity bound in the sediment. Since the determination is based on a competition between 125I-bTSH and the autoantibodies to be determined for common binding sites on the porc. TSHR, all those autoantibodies, and only those autoantibodies, which actually compete with bTSH are detected in this method. Such competing autoantibodies capable of inhibiting the TSH binding are also referred to in the literature as TBII (TBII=thyrotropin-binding inhibitory immunoglobulin), and the extent of their activity is also stated as a percentage of so-called TBII activity.
The competitive radioreceptor assays known to date for the detection of TSH receptor autoantibodies have various disadvantages which are attributable to the quality or availability of the assay components used, to the abnormalities which occur in the sera of individual patients and may falsify the results of the measurements in the known assays and to the fact that the binding capability of TSH receptor preparations is generally very sensitive to changes in the receptor or in the biomolecules bound by it. The binding of biomolecules of a peptide or protein nature, for example hormones or autoantibodies, to receptors is as a rule very complicated, and the formation of a specific bond between receptor and biomolecule is very much more sensitive to structural changes, in particular of the receptor, than is the case with a conventional antigen/antibody binding pair, which forms the basis of most immunoassays in which receptors do not play a role. Attempts to immobilize and/or to label the TSH receptor have to date generally led to structural changes which greatly impair the functionality of the receptor. As a consequence of this, there have to date been scarcely any reworkable descriptions of a practical implementation of numerous basic assay types which are available in immunoassays with the use of antibody/antigen binding for the case of receptor binding assays for the determination of TSHR-auto-ab, so that such other assay types cannot yet be used in practice for the TSHR-auto-ab determination. This applies in particular to those assay types in which immobilized binding partners are employed and the concentration of a tracer bound to a solid phase is determined directly at the end of the measurement, or in which bulky molecules such as enzymes, enzyme substrates or a chemiluminescent label, are used for labelling. Since the measurement of a tracer bound to a solid phase is the basis of most automatic assay apparatuses for series measurements, the known assays for the determination of TSH receptor autoantibodies have not to date been feasible on such automatic apparatuses.
The patent DE 43 28 070 C1 describes a type of receptor binding assay which is based on the coated tube technique and in which the difficulty of the preparation of labelled or immobilized functional receptor preparations is circumvented by binding to the solid phase components of a competing reaction system which to some extent represents a “shadow” of the actual receptor binding reaction. However, the disclosed principle of the method has proved too complicated and therefore not very practicable for providing assays for routine clinical diagnosis. The general statement in said patent on the problem of receptor binding assays in general and of those for the determination of TSH receptor autoantibodies in particular are hereby expressly incorporated by reference.
EP-B-0 488 170 discloses cell-free receptor binding tests in which recombinant fusion receptors comprising an amino-terminal receptor protein and a carrier protein, in particular the constant part (Fc) of the heavy chain of an immunoglobulin, are used, which fusion receptors have been coupled to a solid phase by means of an antiserum or of a monoclonal antibody. The receptors discussed do not belong to the class of the high molecular weight G-protein-coupled glycoprotein receptors. Furthermore, immobilization by binding a carrier protein which is the Fc part of an immunoglobulin is not very suitable for receptor binding assays with the aid of which autoantibodies are to be determined, since the autoantibodies themselves belong to the immunoglobulins and can bind to the immobilization system.
Certain disadvantages of the conventional assays are also associated with the fact that reactants of different animal origins, i.e. stabilized porcine TSHR preparations in combination with labelled bTSH, were used for the determination of human TSHR-auto-ab. It is true that said assay components are distinguished by good mutual binding and permit detection of, for example, about 80% to 90% of the human TSHR-auto-ab occurring in Graves' disease. However, the clinical value which is reduced compared with 100% detection of the TSHR-auto-ab to be detected is presumably also due at least partly to the fact that the autoantibodies occurring in patients' sera are directed against the human TSHR but are determined on the basis of their binding to a porcine TSHR preparation. In spite of the basic availability of human TSHR preparations produced by a recombinant method (rhTSHR), such rhTSHR have not yet been used in clinical assays since any expected advantages were cancelled out by numerous new practical disadvantages. In particular, there has to date been just as little possibility as in the case of the porc. TSHR of using a functional, natural rhTSHR in assays in a form bound to a solid phase (immobilized) or in labelled form.
The publications W. B. Minich, M. Behr and U. Loos, Exp Clin Endocrinol Diabetes 105, 282–290 (1997) and “70th Annual Meeting of the American Thyroid Association”, 15.19.10.1997, Abstract No. 89, and W. B. Minich, J. D. Weymayer, U. Loos, Thyroid, Vol. 8, 3–7 (1998) (in print) disclose that it is possible to immobilize a recombinant human fusion TSHR by means of a peptide residue attached by genetic engineering and to use said fusion TSHR in this form in TSHR-auto-ab determinations. A corresponding summarizing disclosure is also to be found in the non-prior-published International Patent Furthermore, EP-A 0 719 858 describes a method for the preparation of a functional rhTSHR with the aid of a myeloma cell line. The application mentions in general, speculative form the possibility of producing monoclonal antibodies using the rhTSHR polypeptide prepared, and proposes using such antibodies inter alia for immobilizing rhTSHR and making use thereof in the determination of TSHR-auto-ab. However, neither the actual preparation and selection of such monoclonal antibodies are described, nor is it shown concretely that the proposal actually makes it possible to immobilize the rhTSHR without loss of functionality with the optionally obtainable monoclonal antibodies and to use it in this form in the determination of TSHR-auto-ab. The disclosure of EP-A 719 858 is to this extent not reworkable. Monoclonal antibodies are not mentioned in the parent scientific publication (Matsuba et al., J. Biochem. 118, pages 265–270 (1995)).
After the molecular structure of the TSH receptor had been determined, monoclonal and polyclonal antibodies against complete rhTSHR polypeptides, against the N-terminal extracellular part (comprising 398 amino acids without the signal peptide) of such receptors and against conjugates of shorter receptor peptide partial sequences were prepared by numerous working groups with the aim of determining the TSH receptor epitopes responsible for the TSH binding and the antibody binding (cf. for example N. G. Morgenthaler et al., J Clin Endocrinol Metab 81: 3155–3161 (1996); Seetharamaiah G S et al., Endocrinology 134, No. 2, pages 549–554 (1994); Desai R K et al., J. Clin. Endocrinol. Metab. 77: 658–663, 1993; Dallas J S et al., Endocrinology 134, No. 3, pages 1437–1445 (1994); Johnstone A P et al., Mol. Cell. Endocrinol. 105 (1994), R1–R9; Seetharamaiah G S et al., Endocrinology 136, No. 7, pages 2817–2824 (1995); Nicholson L B et al., J. Mol. Endocrinol. (1996) 16, pages 159–170; Ropars A et al., Cell. Immunol. 161, pages 262–269 (1995); Ohmori M et al., Biochem. Biophys. Res. Commun. 174, No. 1 (1991), pages 399–403; Endo T. et al., Biochem. Biophys. Res. Commun. 181, No. 3 (1991), pages 1035–1041; Costagliola S et al., Endocrinology 128, No. 3, pages 1555–1562, 1991; Marion S et al., Endocrinology 130, No. 2, pages 967–975 (1992); J. Sanders et al., J. Endocrinol. Invest. 19 (Suppl. No. 6); 1996, and further publications cited in said publications). The various antibodies were tested for their binding behaviour with respect to the TSH receptor or partial sequences thereof produced by a recombinant method and in particular with respect to their ability to interfere with the binding of TSH to various forms or fragments of the TSH receptor. Since the various polyclonal or monoclonal antibodies had been produced by immunization of different animals and/or with the use of recombinant material from different expression systems and in addition TSH receptors produced by recombinant methods and of various origins or peptide fragments thereof were frequently used in the binding tests, and since furthermore it was found that the glycolization and/or correct folding of the receptor peptide was likely to be decisive for the binding of numerous antibodies, the epitope structure of natural TSH receptors and the epitope-specific binding behaviour of the autoantibodies occurring the polyclonal antibody populations of human sera have not yet been fully determined.
In the publication by Dallas J S et al., Endocrinology 134, No. 3, pages 1437–1445 (1994), for example, a partial recombinant TSH receptor which is prepared with the aid of the Baculovirus/insect cell expression system and has the amino acids of the extracellular domain of the human TSH receptor without the N-terminal signal peptide is used for immunizing rabbits, and specific antibody fractions are obtained from the resulting immunoglobulin fractions by affinity chromatography with the use of synthetic peptides each having about 20 amino acids. Said specific antibody fractions are then investigated, inter alia, with regard to their suitability for blocking the binding of TSH to a solubilized porcine TSH receptor in a commercial receptor binding assay. The antibodies showed no stimulatory activity.
The publication by Seetharamaiah G S et al., Endocrinology 136, No. 7, pages 2817–2824 (1995) describes how the same partial recombinant TSH receptor as in the above publication was used for immunizing mice and for preparing monoclonal antibodies against individual epitopes of the TSH receptor by standard techniques. A similar procedure is described in Nicholson L B et al., J. Mol. Endocrinol. (1996) 16, pages 159–170.
According to Seetharamaiah G S et al., Endocrinology 134, No. 2, pages 549–554 (1994), a partial recombinant TSH receptor prepared as above is subsequently folded and then tested with respect to its suitability for binding radiolabelled bTSH. For this purpose, it is reacted in the liquid phase with radiolabelled bTSH. To separate the resulting complex as quantitatively as possible from the reaction mixture, an antibody which was produced by immunizing rabbits with a conjugate of a partial peptide, containing the amino acids 357 to 372 of the complete TSH receptor sequence, and had been found not to inhibit the binding of bTSH to the unfolded partial recombinant TSH receptor is then added as part of a precipitation system (Desai R K et al., J. Clin. Endocrinol. Metab. 77: 658–663, 1993). The added antibody or the complexes which contained it and bound radiolabelled bTSH is or are then precipitated with the aid of protein A, which binds unspecifically to every antibody. Under the experimental conditions, the binding of protein A to the receptor-bound antibody does not appear to impair the simultaneous bTSH binding.
In the earlier, non-prior-published Patent Application 196 51 093.7, a practically feasible competitive solid-phase receptor assay for the determination of TSHR-auto-ab is furthermore described for the first time, in which assay the TSHR-auto-ab to be determined and labelled bTSH or optionally also a labelled monoclonal antibody are allowed to compete for binding sites of a solubilized TSHR, and the TSHR complexes formed are bound to a solid phase by means of an immobilized monoclonal antibody. In the Application 196 51 093.7, those monoclonal antibodies which recognize relatively short amino acid sequences of the TSHR are used in combination with crude solubilized porcine or optionally also recombinant TSHR preparations. The immobilization of the TSHR complexes is effected in the embodiment with the aid of an affinity gel to which a sequential monoclonal anti-hTSHR-mab has been bound. The focal point of the teaching of the Application 196 51 093.7 is the increase in the clinical value of the TSHR-auto-ab determination, in particular with the use of the conventional crude solubilized TSHR preparations. However, the content of said Application, in particular concerning possible assay variations, is hereby expressly incorporated by reference.
A particular method for obtaining anti-hTSHR-mab is furthermore described by S. Costagliola and G. Vassart in J. Endocrinol. Invest. 20 (Suppl. to no. 5), Abstract 4 (1997). According to this method, immunization of mice for the purpose of antibody formation is effected not with a peptide antigen but by intramuscular injection of a DNA plasmid construct coding for the hTSHR. In this way, novel monoclonal antibodies having high affinity to the natural hTSHR are obtained. This technique has proved very valuable and is also used in the present application, in particular for the preparation of anti-hTSHR-mab which recognize conformational epitopes.
In addition, it may be noted that the knowledge obtained with antibodies against recombinant TSH receptors or parts thereof led to the proposal to use, for the determination of receptor autoantibodies, a third principle, known per se, in the form of an immunoprecipitation assay, in which a preparation of an extracellular part of a recombinant TSH receptor labelled by incorporation of 35S-methionine is used as a reagent for the precipitation. In one such assay, there is no selectivity either for TSI or for TBII (N. G. Morgenthaler et al., J Clin Endocrinol Metab 81: 700–706 (1996)). However, the preparation of the 35S-labelled receptor by in vitro translation is extraordinarily complicated and expensive, and there are no measuring apparatuses which are suitable for a routine clinical measurement of the radiation emitted by 35S. The method is therefore unsuitable as a method of measurement for routine clinical diagnosis.